JP2016519928A - Potato cultivar E12 - Google Patents

Abstract

A potato cultivar termed E12 is disclosed. The present invention relates to tubers of potato cultivar E12, seeds of potato cultivar E12, plants and plant parts of potato cultivar E12, food products produced from potato cultivar E12, and potato cultivar E12 itself or separately. Relates to a method for producing potato plants by crossing with potato varieties. The present invention also relates to methods for producing transgenic potato plants, as well as transgenic potato plants and parts thereof produced by these methods.

Description

(Cross-reference of related applications)
This application claims priority to US Provisional Patent Application No. 61 / 818,752 (filed May 2, 2013), the entirety of which is incorporated herein by reference.

  The present invention relates to a novel potato cultivar named E12, and tubers, plants, plant parts, cultured tissues and seeds produced by the potato varieties. The present invention further relates to food products derived from potato cultivar E12, such as French fries, potato flakes and potato granules. All publications mentioned in this application are herein incorporated by reference.

Potato is the fourth most important food harvest in the world and by far the most important vegetable. Potatoes are currently raised for commercial use in almost all states in the United States. Annual potato production exceeds 1.6 × 10 ¹⁰ kg (18 million tons) in the United States and over 2.72 × 10 ¹¹ kg (300 million tons) worldwide. The popularity of potatoes is mainly due to its versatility and nutritional value. The potatoes can be used fresh, frozen or dried, or processed into powder, starch or alcohol. These contain carbohydrate complexes and are rich in calcium, niacin and vitamin C.

  Potato quality works badly in the food industry due to two significant factors: (1) Asparagine, a non-essential amino acid that is rapidly oxidized when fried or baked to form acrylamide, a carcinogen And (2) potatoes are very susceptible to enzymatic browning and discoloration, an undesirable event that occurs when polyphenol oxidase leaks from a damaged potato's damaged plastid. In the cytoplasm, the enzyme oxidizes phenol, which rapidly polymerizes to produce dark pigments. Tubers contain large amounts of phosphorylated starch, some of which are broken down during storage to produce glucose and fructose. These reducing sugars react with amino acids to form Maillard products containing acrylamide when heated at temperatures in excess of 120 ° C. The two enzymes involved in starch phosphorylation are water dikinase R1 and phosphorylase-L (R1 and PhL). Browning also occurs non-enzymatically as a result of partial degradation of starch to glucose and fructose.

  Currently, there are no potato plant varieties that produce tubers with low acrylamide content, increased resistance to black spot wounds and reduced aging sweetness. Thus, there is a need to develop potato varieties that have reduced levels of toxic compounds, but without the use of unknown or exogenous nucleic acids. The present invention meets this need.

  The above examples of related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those skilled in the art upon reading this specification.

  The following embodiments and aspects thereof are described in combination with systems, tools and methods, which are meant to be exemplary and not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

  To this end, the present invention is a novel transformant with a nucleic acid sequence that is native to the potato plant genome and does not contain foreign DNA, Agrobacterium DNA, viral markers or vector backbone sequences. Potato variant E12 is provided. Rather, the DNA inserted into the genome of potato variant E12 is native to potato or non-coding native to wild potato, a plant that is reproductively compatible with potato. A polynucleotide that silences genes involved in black spot development, asparagine accumulation, and increased aging sweetness.

  Accordingly, in one embodiment, the present invention provides a plant vector called pSIM278 comprising a first silencing cassette and a second silencing cassette, wherein the first silencing cassette is in the antisense orientation and the asparagine synthetase- 1 gene (fAsn1) fragment and 2 ′ DNA segment of polyphenol oxidase-5 gene 3′-untranslated sequence, second silencing cassette in the antisense orientation, potato phosphorylase-L (pPhL) It contains two copies of DNA, a gene fragment and a potato R1 gene fragment. The pSIM1278 vector is a 9,511 bp backbone region that supports the maintenance of plant DNA prior to plant transformation and does not migrate into plant cells during plant cell transformation, and within the plant cell genome during transformation. A DNA insertion region of 10,147 bp containing native DNA stably integrated into

  In different embodiments, the present invention provides plant cells transformed with the plant vectors of the present invention. In a further embodiment, the present invention provides a potato plant variety comprising one or more cells transformed with the plant vector of the present invention. In one aspect of the invention, the potato plant varieties express at least one of the two silencing cassettes of the vector, and the silencing cassette expression is asparagine synthetase-1 gene and polyphenols in the tuber of the endogenic plant. Provides down-regulation of the oxidase-5 gene. In a preferred aspect of the present invention, tubers of potato plant varieties expressing at least one silencing cassette present two or more desired traits that are not present in the tubers of non-transformed plants of the same varieties. In a most preferred aspect of the invention, the two or more desired traits are selected from the group consisting of low asparagine accumulation, reduced black spot damage and reduced heat-induced acrylamide formation.

  In different aspects of the present invention, the potato plant variant expresses both silencing cassettes of the plant DNA vector, and the silencing cassette expression is expressed in the tubers of the potato plant variant asparagine synthetase-1 gene and polyphenol oxidase-5. It leads to downregulation of the gene, phosphorylase-L gene and dikinase R1 gene. In a preferred aspect of the invention, tubers of potato plant varieties that express two silencing cassettes of a plant DNA vector present two or more desired traits that are not present in the tubers of non-transformed plants of the same variant. In a preferred embodiment, the two or more desired traits are selected from the group consisting of low asparagine accumulation, reduced black spot wounds, reduced reducing sugar accumulation during storage, and reduced heat-induced acrylamide formation. In one aspect of the invention, the potato plant variant that expresses the two silencing cassettes of the plant DNA vector is a Russet Burbank E12 variant.

  Therefore, in accordance with the present invention, Solanum tuberosum L., designated E12. New potato cultivars of the genus and species of (Solanum tuberosum L.) are provided. Therefore, the present invention self-pollinates potato cultivar E12, potato cultivar E12 tuber, potato cultivar E12 plant, potato cultivar E12 seed, potato cultivar E12 food product, and potato cultivar E12. Or a method for producing potato cultivar E12 by crossing potato cultivar E12 with another potato cultivar and producing variants by mutagenesis or transformation of potato cultivar E12.

Thus, any such method using cultivar E12 is an embodiment of the invention: self-pollination, backcrossing, hybrid production, crossbreeding to a population, etc. All plants produced using potato cultivar E12 as at least one parent are within the scope of the present invention. Conveniently, the potato cultivar may be used for crossing with other different potato plants to produce first generation (F ₁ ) potato hybrid tubers, seeds and plants with superior characteristics. .

  In another embodiment, the present invention provides single or multiple gene conversion plants of potato cultivar E12. In one embodiment, the transferred gene may be a dominant allele or a recessive allele. In some embodiments, the transferred gene is herbicide resistance, insect resistance, resistance to bacterial, fungal or viral diseases, male fertility, male sterility, increased nutritional quality, uniformity, and It imparts traits such as increased concentrations of starch and other carbohydrates, reduced ease of scratching and reduced conversion of starch to sugar. This gene may be a naturally occurring potato gene or a transgene introduced through genetic engineering techniques, backcrossing or mutation.

  In another embodiment, the present invention provides a renewable cell for use in tissue culture of potato cultivar E12. In one embodiment, the tissue culture reproduces a plant having all of the physiological and morphological characteristics of the potato plant described above, and a plant having substantially the same genotype as the potato plant described above. It is possible to play. In some embodiments, the renewable cells in such tissue culture are embryos, protoplasts, meristem cells, callus, pollen, leaves, buds, pistils, cotyledons, hypocotyls, roots, root canals, flowers, Seeds, petiole, tubers, buds or stems. Still further, the present invention provides a potato plant regenerated from the tissue culture of the present invention.

  In a further embodiment, the present invention provides a food product made from tubers of the potato plant variety Russet Burbank E12. Preferably, the food product is a heat treated product. Even more preferably, the food product is fried potato, potato chips, dehydrated potato material, potato flakes or potato granules.

  In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent from the study of the description that follows.

The pSIM1278 transformation vector is illustrated. The vector backbone region on the left is 9,511 bp long, starts at 9,957 bp and ends at 19,468 bp. Skeletal DNA consists primarily of bacterial DNA that provides an indication of the maintenance of the DNA insert prior to plant transformation. The DNA insertion region (right side) is 10,147 bp long (19,469 bp to 19,660 bp and 1 bp to 9,956 bp). The DNA insert consisted only of native DNA and was stably integrated into the potato gene upon transformation. FIG. 4 provides a schematic of a silencing cassette in a DNA insert inserted into a pSIM1278 transformation vector. Each silencing cassette contains two copies of two gene fragments separated by a spacer. Two copies of a DNA segment containing fragments of four target genes, Asn-1, Ppo-5, Phl and R1, are inserted as inverted repeats between two convergent promoters denoted as Pro, It is predominantly active in tubers. The resulting plant containing the silencing cassette produces a series of RNA molecules that are diverse and not polyadenylated in the tubers, which dramatically and vigorously silence the intended target gene. The size of the RNA molecule is generally shorter than the distance between the two promoters used. This is because reverse transfer results in collision transfer. The result of a catechol assay is shown. Russet Burbank control is on the left and variant E12 is on the right. Dark brown indicates that there is no reduction in black spots. As shown in FIG. 3, E12 has a trait for reduced black spot, while having no control.

In the following description and tables, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:
Allele. An allele is any one or more alternative forms of a gene for a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

  Amino acid arrangement. As used herein, oligopeptides, peptides, polypeptides or proteins isolated from plants, native to plants, naturally occurring in plants, or made synthetically and Including those fragments, but not including the nucleic acid sequence of the endogenous counterpart.

  It was manipulated artificially. As used herein, “artificially manipulated” moves or places a plant or plant cell by hand, by mechanical means, or by recombinant means such as genetic engineering techniques. Or a plant having a biological, biochemical, morphological, or physiological phenotype and / or genotype that is different from the naturally occurring counterpart that is not manipulated by surgery or control It means producing plant cells.

  Asexual breeding. Producing offspring by creating infectious plants from leaf cuts, stem cuts, root cuts, tuber buds, running roots, single plant cell protoplasts, callus, etc. Not included.

  Skeleton. A binary vector nucleic acid sequence, excluding DNA insertion sequences that are intended to be transferred.

Backcross. Backcrossing, growth type, the process of repeatedly crossing back the hybrid progeny to one parent, for example, refers to the process of the first generation hybrids F ₁ with one mating parental genotypes of F ₁ hybrids.

  Bacterial ring rot. Bacterial ring rot is a disease caused by the bacterium Clavibacter michiganense subspecies. Bacterial ring rot originates from the characteristic collapse of the conduit ring in the tuber. This ring often appears as a cream yellow to light brown, cheese-like rot. On the outer surface of the potato, the heavily infested tubers are slightly wilted, dry and show cracked areas. The signs of bacterial ring rot in the tuber tissue of the infected tubers are less obvious than above, and may appear only as collapsed, sporadic dark lines or as continuous yellowing.

  Black spot. The black spots found in damaged tubers are the result of a pigment called melanin that is produced after cell damage and makes the tissue look brown, gray or black. Melanin is formed when the phenolic substrate and the appropriate enzyme come into contact with each other as a result of cell damage. This damage does not require breaking the cells. However, this mixing of substrate and enzyme usually occurs whenever a tissue collides. The dark spots first occur in the marginal tissue immediately below the conduit ring, but may be large enough to include portions of cortical tissue.

  Boundary-like array. A “boundary-like” sequence is isolated from a plant species selected for modification, or a plant species that is reproductive compatible for modification, and functions similarly to the boundary sequence of Agrobacterium. That is, the border-like sequences of the present invention facilitate and facilitate the incorporation of the polynucleotide to which it is linked. The boundary-like sequence is 5 to 100 bp long, 10 to 80 bp long, 15 to 75 bp long, 15 to 60 bp long, 15 to 50 bp long, 15 to 40 bp long, 15 to 30 bp long, 16 to 30 bp long, 20 to 30 bp long, It is between 21-30 bp length, 22-30 bp length, 23-30 bp length, 24-30 bp length, 25-30 bp length, or 26-30 bp length. The left and right border sequences are isolated from and / or native to the genome of the plant to be modified. The border-like sequence is not identical in nucleotide sequence to any known Agrobacterium-derived T-DNA border sequence. Thus, the border-like sequences are different from T-DNA border sequences derived from Agrobacterium, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes, 1, 2, 3, 4 It may have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. That is, the border sequence or border-like sequence of the present invention comprises at least 95%, at least 90%, at least 80% of a T-DNA border sequence from an Agrobacterium species such as Agrobacterium tumefaciens or Agrobacterium rhizogenes, Has at least 75%, at least 70%, at least 60% or at least 50% sequence identity but no 100% sequence identity. Boundary-like sequences can be isolated from the plant genome and modified or mutagenized to alter the efficiency with which a nucleotide sequence can be incorporated within another nucleotide sequence. Other polynucleotide sequences may be added to or incorporated within the border-like sequences of the present invention. Thus, the left or right border may be modified to have 5'- and 3'-multicloning sites or additional restriction sites. The border sequence may be modified to increase the probability that backbone DNA from the accompanying vector will not integrate into the plant genome.

  Essentially consists of. Compositions “consisting essentially of” certain elements are limited to those elements and elements that do not substantially affect the basic and novel characteristics of the composition of the present invention. Therefore, as long as the composition does not affect the basic and novel features of the present invention, i.e. does not contain exogenous DNA that is not derived from the selected plant species or reproductive compatible with the selected plant species. This composition may be considered a component of the composition of the present invention, i.e. characterized by the word "consisting essentially of".

  Cotyledon. Cotyledons are a type of seed leaf. The cotyledon contains seed food storage tissue.

  Degenerate primer. A “degenerate primer” is an oligonucleotide that contains sufficient oligonucleotide variation that can accept base mismatches when hybridizing to similar but not identical homologous sequences.

  Dicotyledonous plant (dicot). A flowering plant in which the embryo has two leaves or cotyledons. Examples of dicot include legumes including tobacco, tomato, potato, sweet potato, cassava, alfalfa and soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, pumpkin, daisies and cactus. However, it is not limited to these.

  DNA insert. According to the present invention, a DNA insert that is inserted into the genome of a plant contains a polynucleotide sequence that is native to the plant or has genetic elements that are native to the plant. In one example, for example, the DNA insert of the potato variant E12 of the present invention is a 10,147 bp non-coding polynucleotide native to wild potato, a potato or potato reproductive compatible plant, upon transformation. It is stably integrated into the genome of plant cells to silence genes involved in black spot development, asparagine accumulation and increased aging sweetness. The DNA insert preferably contains two expression cassettes and is inserted into a transformation vector called the pSIM1278 transformation vector. The first cassette is a fragment of both the asparagine synthetase-1 gene (Asn1) and the polyphenol oxidase-5 gene (Ppo5), the Agp promoter of the ADP glucose phosphorylase gene (Agp) and the Gbss promoter of the granule-bound synthase gene (Gbss). Including and including as an inverted repeat. These promoters are predominantly active in tubers. The function of the second cassette is to silence the promoters of the starch-related gene dikinase-R1 (R1) and the phosphorylase-L gene (PhL). This cassette consists of promoter fragments of starch-related gene dikinase-R1 (R1) and phosphorylase-L gene (PhL) operably linked to the same Agp and Gbss promoters as the first cassette. These expression cassettes do not contain foreign genes and consist only of DNA from either the selected plant species or plants that are reproductive compatible with the selected plant species.

  Embryo. Embryos are immature plants contained within mature seeds.

  Exogenous. “Exogenous” refers to a nucleic acid that is, in terms of nucleic acid, derived from a non-plant organism, or derived from a plant that is not the same as the transformed plant, or is not crossable with the transformed plant. It is not derived and means not belonging to the target plant. In accordance with the present invention, exogenous DNA or RNA refers to a nucleic acid that is naturally present in the genetic nature of a fungus, bacterium, virus, mammal, fish or bird, but not naturally present in the transformed plant. Thus, an exogenous nucleic acid encodes a polypeptide that is not naturally produced by, for example, a transformed plant. The exogenous nucleic acid need not encode a protein product. According to the present invention, the desired genetic plant is one that does not contain any foreign nucleic acid in its genome.

  gene. As used herein, “gene” refers to a coding region and does not include nucleotide sequences that are 5′- or 3′- of this region. A functional gene is a coding region operably linked to a promoter or terminator. Genes can be introduced into the genome of any species of different species or the same species using transformation or various propagation methods.

  The gene was converted. A (transformed) genetically transformed (transformed) plant refers to a plant developed by a plant propagation technique called backcrossing, where essentially all of the desired morphological and physiological characteristics of the varieties are It has been recovered in addition to one or more genes transferred via backcrossing techniques, genetic engineering or via mutations into the variant. One or more loci are also imported.

  Genetic rearrangement. Refers to the reassembly of genetic elements that can occur spontaneously in vivo as well as in vitro, introducing a new composition of genetic material. For example, splicing together polynucleotides at different loci can occur spontaneously in vivo during both plant development and reproductive recombination. Thus, recombination of genetic elements in vitro by non-natural genetic modification techniques approximates recombination events that can occur through reproductive recombination in vivo. Insertion of a DNA insert into the plant genome is an example of gene rearrangement or genome rearrangement, for example.

  Potato cyst nematode (Golden nematode). Globodera rostochiensis, commonly known as potato cyst nematode, is a plant parasitic nematode that affects the roots and tubers of potato plants. Signs include poor plant growth, wilting, water stress and poor nutrition.

  Hypocotyl. The hypocotyl is the part of the embryo or seedling between the cotyledon and the root. This can therefore be considered as a transition zone between shoots and roots.

  Inside the frame. Nucleotide triplets (codons) are translated into the adjacent amino acid sequence of the desired recombinant protein in plant cells. Specifically, the present invention contemplates a first nucleic acid linked in a reading frame to a second nucleic acid, wherein the first nucleotide sequence is a gene and the second nucleotide sequence is a promoter or Similar regulatory elements.

  Built-in. The insertion of a nucleic acid sequence derived from a selected plant species or a plant that is reproductive compatible with the selected plant species into the genome of the cell of the selected plant species. “Integration” refers to the integration of native genetic elements into the phagocytic cell genome. In order to incorporate native genetic elements, for example by homologous recombination, the present invention may “use” non-native DNA as a step in such a process. Thus, the present invention distinguishes between “use” of a particular DNA molecule and “integration” of a particular DNA molecule into the plant cell genome.

  Introduction. As used herein, refers to the insertion of nucleic acid sequences into cells by methods that include infection, transfection, transformation or transfection.

  Isolated. “Isolated” refers to a nucleic acid or compound that is physically separated from its normal native environment. Isolated material may be maintained in a suitable solution, including, for example, solvents, buffers, ions, or other components, and may be in pure or non-pure form.

  leader. A sequence that precedes a gene (ie, is 5 ') and is transcribed but not translated.

  Locus. A locus is one or more traits such as male sterility, herbicide resistance, insect resistance, disease resistance, waxy starch, modified fatty acid metabolism, modified phytic acid metabolism, modified carbohydrate metabolism and modified Confer protein metabolism. This trait can be confirmed, for example, by a naturally occurring gene introduced into the genome of this variant by backcrossing, natural or induced mutations, or by a transgene introduced through genetic transformation techniques. A locus may contain one or more alleles integrated at one chromosomal location.

  Yield for market. Market yield refers to the weight of all tubers harvested between 2 and 4 inches in diameter. Market yield is measured at cwt = 45 kg (100 pounds) cwt (100 weight).

  Monocot. A flowering plant in which the embryo has one leaf or cotyledon. Examples of monocot include turfgrass, corn, rice, oats, wheat, barley, sorghum, orchid, iris, lily, onion and palm.

  Native. A “native” genetic element refers to a nucleic acid that is naturally occurring in, derived from, or belonging to the genome of the plant to be transformed. Thus, any nucleic acid, gene isolated from the genome of the plant or plant species to be transformed or detected from a plant or plant species that is reproductive compatible with the transformed plant species, ie, capable of mating A molecule of polynucleotide, DNA, RNA, mRNA or cDNA is “native”, ie unique, to this plant species. In other words, native genetic elements represent all genetic material that is accessible to plant growth types for plant improvement through classical plant propagation. Any variant of a native nucleic acid is also considered “native” in accordance with the present invention. In this regard, a “native” nucleic acid may also be isolated from a plant or its reproductive compatible species, or the resulting variant is relative to an unmodified and native nucleic acid isolated from a plant. 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85% in nucleotide sequence 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68% , 67%, 66%, 65%, 64%, 63%, 62%, 61% or 60% or more may be modified or mutated. Native nucleic acid variants may also be less than about 60%, about 55%, or less than about 50% similar in nucleotide sequence. A “native” nucleic acid isolated from a plant may also encode a variant of a naturally occurring protein product that has been transcribed and translated from the nucleic acid. Thus, a “native” nucleic acid is also 99%, 98%, 97%, 96%, 95%, 94% relative to the amino acid sequence of the unmodified and native protein expressed in the plant from which the nucleic acid was isolated. 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77 %, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or It may encode proteins that are 60% or more similar.

  Native genetic element. A “native genetic element” can be incorporated and incorporated into the genome of a selected plant species according to the present invention. Native genetic elements are isolated from plants belonging to the selected plant species or plants that are reproductive compatible with the selected plant species. For example, the native DNA incorporated into cultivated potato (Solanum tuberosum) is S. cerevisiae. It can be derived from any genotype of tuberosam or S. cerevisiae. It may be derived from any genotype of a wild potato species that is reproductive compatible with tuberosam (eg, S. demissum).

  A naturally occurring nucleic acid. Naturally occurring nucleic acids are found in the genome of the selected plant species and may be DNA molecules or RNA molecules. Restriction site sequences that normally exist within the genome of a plant species can be genetically engineered in exogenous DNA molecules, such as vectors or oligonucleotides, even if the restriction sites are not physically isolated from the genome. Thus, the present invention provides for the synthetic creation of a nucleotide sequence, such as a restriction enzyme recognition site, within the genome of a selected plant species to be transformed or a plant that is reproductive compatible with the selected plant species. Accept as long as it exists in nature.

  Operatively linked. Combining two or more molecules in such a way that they function properly in combination in plant cells. For example, a promoter is operably linked to a structural gene when the promoter controls transcription of the structural gene.

  As used herein, the term `` plant '' refers to angiosperms and gymnosperms such as potato, tomato, tobacco, alfalfa, lettuce, carrot, strawberry, sugar beet, cassava, sweet potato, soybean, corn, turf Examples include, but are not limited to, glass, wheat, rice, barley, sorghum, oats, oak, eucalyptus, walnut and palm. Therefore, the plant may be a monocot or a dicot. The term “plant” as used herein, whether produced by reproduction or non-reproductively, is a plant cell, seed, plant progeny, propagule, as well as any product thereof, For example, cuts or seeds are also included. Plant cells include suspension cultures, callus, embryos, growth point regions, callus tissues, leaves, roots, shoots, gametophytes, spores, pollen, seeds and microspores. Plants can be at various stages of maturity and can be grown in pots, greenhouses or outdoors, in liquid or solid culture, or grown in soil or in a suitable medium. Expression of the introduced leader, trailer or gene sequence in the plant may be temporary or permanent. The “selected plant species” can be, but is not limited to, any one of these “plants”.

  Plant part. As used herein, the term “plant part” (or potato plant or part thereof) includes protoplasts, leaves, stems, root crowns, cocoons, pistils, seeds, embryos, pollen, ovules, cotyledons, hypocotyls. , Flowers, tubers, buds, tissues, petioles, cells, meristem cells, and the like.

  Plant species. A group of plants belonging to various publicly named plant species that exhibit at least some reproductive compatibility.

  Plant transformation and cell culture. Broadening refers to the process by which plant cells are genetically modified and transferred to a suitable plant culture medium for maintenance, further growth, and / or further development.

  Proper breeding. An individual of a native gene and a regulatory element isolated from a nucleic acid, for example from a selected plant species or from another plant of the same species as the selected plant or from a species that is reproductive compatible with the selected plant species Refers to the improvement of the plant by the stable introduction of plant cells into the plant cells and the subsequent regeneration of these genetically modified plant cells throughout the plant. Since unknown or exogenous nucleic acids are not permanently integrated into the plant genome, the technology of the present invention uses the same genetic material that is also available through traditional plant propagation.

progeny. As used in this document, at least one of the plants comprises F ₁ potato plants produced from a cross of two potato plants comprising potato cultivars E12, progeny further intergenerational mating with regression and parent strains Subsequent F ₂ , F ₃ , F ₄ , F ₅ , F ₆ , F ₇ , F ₈ , F ₉ and F ₁₀ are included, but are not limited to.

  Quantitative trait locus (QTL). Quantitative trait loci (QTLs) refer to loci that control traits that are normally continuously distributed and that can be represented numerically to some extent.

  Recombinant. As used herein, a wide variety of techniques are described, in which genes can be cloned, DNA can be sequenced, and protein products can be produced. As used herein, the term also describes proteins that are produced after transfer of the gene into cells of the plant host system.

  Regeneration. Regeneration refers to the generation of plants from tissue culture.

  Regulatory sequence. Standard and known to those skilled in the art that can be included in an expression vector to increase and / or maximize transcription of a gene of interest or translation of the resulting RNA in a plant system. These include, but are not limited to, promoters, peptide transport signal sequences, introns, polyadenylation, and transcription termination sites. Methods for modifying nucleic acid constructs to increase expression levels in plants are also known in the art (eg, Rogers et al., 260 J. Biol. Chem. 3731-38, 1985; Cornejo et al., 23). See Plant Mol.Biol.567: 81, 1993). Various factors are known in the art for the manipulation of plant systems to affect the rate of protein transcription, including regulatory sequences such as enhancers and silencers that act positively or negatively. As well as the chromosomal structure can have an effect. The present invention presents that at least one of these factors can be utilized in manipulating plants to express the protein of interest. The regulatory sequences of the present invention are native genetic elements, i.e. isolated from the plant species selected to be modified.

  Selectable marker. A “selectable marker” is a gene that typically encodes a protein that confers a certain resistance to antibiotics, herbicides or toxic compounds and is used to identify transformation events. Examples of selectable markers include streptomycin phosphotransferase (spt) gene encoding streptomycin resistance, phosphomannose isomerase (pmi) gene converting mannose-6-phosphate to fructose-6 phosphate, encoding resistance to kanamycin and geneticin Inhibits the action of neomycin phosphotransferase (nptII) gene, hygromycin phosphotransferase (hpt or aphiv) gene encoding resistance to hygromycin, acetolactate synthase (als) gene encoding resistance to sulfonylurea herbicides, glutamine synthase Genes encoding resistance to herbicides that act like phosphinotricin or bas a (e.g., bar gene), or others known similar gene in the art.

  Sense suppression. Reduction of endogenous gene expression by expression of one or more additional copies of all or part of the gene in the transgenic plant.

  specific gravity. As used herein, “specific gravity” is a representation of density and a measure of potato quality. There is a high correlation between tuber specific gravity and starch content and the percentage of dry matter or total solids. High specific gravity contributes to a higher percentage of processed products and better quality.

  T-DNA-like. A “T-DNA-like” sequence is a nucleic acid isolated from a selected plant species or from a plant that is reproductive compatible with the selected plant species, and is at least 75% relative to the Agrobacterium species T-DNA Share 80%, 85%, 90% or 95% sequence identity but not 100%. A T-DNA-like sequence can include one or more border sequences or border-like sequences, each of which can incorporate a nucleotide sequence into another polynucleotide.

  Total yield. Total yield refers to the total weight of all harvested tubers.

  Trailer. A sequence that is transcribed but not translated following the gene (ie, 3 ').

  Transcribed DNA. DNA that contains both the gene and the untranslated leader and trailer associated with the gene, which is transcribed as a single mRNA by the action of the preceding promoter.

  Plant cell transformation. The process by which DNA is stably integrated into the genome of plant cells. “Stable” refers to the permanent or non-transient retention and / or expression of a polynucleotide by the cell genome into the cell genome. Thus, a stably integrated polynucleotide is a fixative within the transformed cell genome and can be replicated and propagated through continuous progeny of the cell or the resulting transformed plant. Transformation can occur under artificial conditions using natural conditions or various methods well known in the art. Transformation can depend on any known method for insertion of nucleic acid sequences into prokaryotic or eukaryotic host cells, including Agrobacterium-mediated transformation protocols, viral infections, whisker methods, electro Examples include poration, heat shock, lipofection, polyethylene glycol treatment, microinjection and particle gun.

  Transgene. A gene inserted into the host genome containing the protein coding region. In the context of the present invention, the element comprising the transgene is isolated from the host genome.

  Transgenic plant. A genetically modified plant comprising at least one transgene.

  variant. As used herein, “variant” is understood to mean a nucleotide or amino acid sequence that deviates from the standard or predetermined nucleotide or amino acid sequence of a particular gene or protein. The terms “isoform”, “isoform” and “analog” also refer to “variant” forms of nucleotide or amino acid sequences. An amino acid sequence that has been modified by the addition, removal or substitution of one or more amino acids, or a change in nucleotide sequence can be considered a “variant” sequence. A variant may have “conservative” changes in which the substituted amino acid has similar structural or chemical properties, eg, replacement of leucine with isoleucine. A variant may have “nonconservative” changes, eg, replacement of glycine with tryptophan. Similar minor variations can also include amino acid deletions or insertions, or both. Guidance in determining whether amino acid residues can be substituted, inserted, or deleted can be found using computer programs well known in the art, such as Vector NTI Suite (InforMax, MD) software. .

  Vine aging. Vine ripening refers to the plant's ability to continue to utilize carbohydrates and photosynthesize. Vine ripening is scored on a scale of 1-5, where 1 = dead vine and 5 = vine green, still flowering.

  Inserting the desired trait into the genome of a potato plant presents particular difficulties. This is because potato is tetraploid, highly heterozygous, and sensitive to inbreeding depression. Therefore, it is very difficult to efficiently develop transgenic potato plants that produce less acrylamide and fewer harmful Maillard reaction products during processing using conventional breeding. Harmful Maillard reaction products include the following: N-nitroso-N- (3-keto-1,2-butanediol) -3′-nitrotyramine (Wang et al., Arch Toxicol 70: 10-5 , 1995), 5-hydroxymethyl-2-furfuranyl (Janzowski et al., Food Chem Toxicol 38: 801-9, 2000), and other Maillard reaction products due to mutagenic properties (Shibamoto, Prog Clin Biol Res 304: 359-76, 1989).

  Several methods have been tested and research is ongoing to reduce acrylamide through processing changes, dextrose reduction, and additives such as asparaginase, citrate and competing amino acids. The major expenditure required to implement processing changes through the potato industry is in the millions of dollars. In addition to spending, these processing changes have important drawbacks, including the potentially negative flavor associated with additives such as asparaginase or citrate. Typically, frying manufacture adds dextrose to develop the desired golden brown color during the processing of french fries, but dextrose also increases acrylamide formation through the Maillard reaction. A significant reduction in acrylamide is mainly brought about by omitting dextrose from processing, but the golden brown sign must be developed in some other way (eg through the addition of a dye such as Anato). The use of alternative pigments results in the absence of typical flavors emanating through these browning reactions. A further challenge with the use of additives to reduce reactants such as asparagine is the moisture transfer that occurs during cryopreservation, which results in a resurgence of asparagine to the surface and an increase in acrylamide. Finally, the blackening that occurs after the potato has been damaged affects the quality and recovery during processing of french fries and potato chips. Damaged and damaged potatoes must be scraped or removed prior to processing, resulting in quality challenges or economic losses.

  The “native technology” strategy of the present invention is the expression of polyphenol oxidase-5 (PPO-5) responsible for black spot, asparagine synthetase-1 (Asn-1) responsible for accumulation of asparagine, which is a precursor of acrylamide formation And / or the expression of phosphorylase-L and kinase-R1, which are enzymes involved in the accumulation of reducing sugars that react with amino acids, such as asparagine, to form toxic Maillard products that normally contain acrylamide. To direct the demand of the potato industry to improve the agricultural characteristics and nutritional value of the potato. Partial or complete silencing of these genes in tubers reduces the possibility of producing acrylamide. The use of the native technology of the present invention is no exogenous, only by “native” genetic material that contains only non-coding regulatory regions, which is genetic material obtained from potato plants or plants that are reproductive compatible with potato plants. Transformation of potatoes without integrating any genetic material into the plant genome allows the integration of the desired trait into the genome of a marketable potato plant variety. Desired traits are high resistance to impact-induced black spots, reduced formation of the acrylamide precursor asparagine and reduced reducing sugar accumulation, and consequently reduced accumulation of Maillard products including acrylamide Including improved quality as well as food color control. Integration of these desired traits into existing potato varieties is impossible to achieve through conventional breeding. This is because potato is tetraploid, highly heterozygous, and sensitive to inbreeding depression.

  The non-coding potato plant DNA insert used in the present invention is native to the potato plant genome and does not contain any Agrobacterium DNA. The DNA insert preferably contains two expression cassettes and is inserted into a transformation vector called the pSIM1278 transformation vector. The first cassette is a fragment of both the asparagine synthetase-1 gene (Asn1) and the polyphenol oxidase-5 gene (Ppo5), the Agp promoter of the ADP glucose phosphorylase gene (Agp) and the Gbss promoter of the granule-bound synthase gene (Gbss). Including and including as an inverted repeat. These promoters are predominantly active in tubers. The function of the second cassette is to silence the promoters of the starch-related gene dikinase-R1 (R1) and the phosphorylase-L gene (PhL). This cassette consists of promoter fragments of starch-related gene dikinase-R1 (R1) and phosphorylase-L gene (PhL) operably linked to the same Agp and Gbss promoters as the first cassette. These expression cassettes do not contain foreign genes and consist only of DNA from either the selected plant species or plants that are reproductive compatible with the selected plant species.

  A commercially valuable potato plant variety used in the present invention is Russset Burbank. Luther Burbank developed this variety in the early 1870s. Plants grow well and have an indeterminate type of growth. The trunk is thick, has a splay angle, and has fine spots. The leaves are long to medium width and light to medium green. The flowers are slight, white and sterile. The cultivars are resistant to fruit rot, but are susceptible to Fusarium and Verticillium wilt, cigar and net necrosis, and viruses. Plants require high and uniform soil moisture and controlled nitrogen fertility conditions to produce tubers that do not have bumps, cusps and array projections. When plants are exposed to stress, they generate jelly ends and sugar ends. The tubers produced have a large brown skin and white content, exhibit good long-term storage characteristics and represent a standard for excellent baking and processing quality. The Russet Burbank variant is more likely to develop black spots, has a high free asparagine content and a high ability to increase aging sweetness (Am. J. Potato Res (1966) 43: 305-314). This variety is fertile and is widely bred in the northwest and midwest, especially for the production of french fries.

  The present invention provides a remarkable market value potato variant, namely Russet Burbank, which has been transformed and successfully grown by the transformation vector pSIM1278 identified using the polymerase chain reaction rather than a marker. Also provided is a manufactured food product of the tuber of the potato plant variant E12 of the present invention. Potato cultivar E12 has the following unique plant variant identifier by the Organization for Economic Co-operation and Development (OECD): SPS-φφE12-8.

  Targeted gene silencing with native DNA reduces the level of RNA transcripts of the targeted gene in tubers of potato plant variety E12. Asn1 and Ppo5 gene silencing is sufficient to significantly reduce acrylamide formation by a factor of 2 to 4 without further inhibition of starch-related gene kinase-R1 (R1) and phosphorylase-L (PhL). Accordingly, the tuber of the in-genic potato plant variant E12 of the present invention incorporates a highly desirable trait including a reduced ratio of free amino acids asparagine and glutamine, which is associated with reduced acrylamide formation when fried or baked. Specifically, the potato varieties E12 of the present invention are characterized by a reduction of the asparagine content by less than 2 to 4 times and a reduction in discoloration associated with black spots. Furthermore, the potato cultivar E12 of the present invention shows a delayed degradation of starch into glucose and fructose during storage. Failure to convert starch to sugar further reduces aging sweetness and acrylamide formation, limiting heat-induced browning.

  The potato varieties E12 of the present invention are therefore very popular in the potato industry and the food market because their tubers produce significantly less acrylamide during heat treatment and do not have any potentially harmful foreign genes. High value.

  The present invention uses native technology to integrate native non-coding DNA into the genome of selected potato plant varieties and to develop new intragenic potato plant varieties. The method includes novel identification that includes trait identification, vector design, vector integration into Agrobacterium, selection of recipient potato varieties, plant transformation, illumination in the absence of an open reading frame, and native DNA only. Includes confirmation of potato varieties. Potato cultivar E12 of the present invention is less likely to form acrylamide, has a lower amount of sucrose, and is more resistant to black spots than its non-transformed counterpart.

Example 1. pSIM1278 Transformation Vector The transformation vector pSIM1278 used in the present invention is derived from pSIM106, from which a 0.4-kb potato plant DNA fragment (deposited as GenBank accession number AY567555) is derived from plasmids pVS1 and pBR322. And was ligated to a 5.9-kb SacII-SphI fragment of pCAMBIA1301 (CAMBIA, Canberra, Australia) with the nptIII gene for bacterial resistance to kanamycin. An expression cassette containing the Agrobacterium ipt gene and the preceding Ubi-3 promoter (Garbarino and Belknap, 1994) and the following Ubi-3 terminator was introduced into the vector backbone as a 2.6-kb SacII fragment ( Rommens et al., 2004). Insertion of a native 10-kb DNA segment with two silencing cassettes into the DNA insert of pSIM106 resulted in pSIM1278. This vector was used for all transformations. The pSIM1278 vector map is shown in FIG. The vector skeleton region is 9,511 bp, starting at a position of 9,957 bp and ending at a position of 19,468 bp. Skeletal DNA consists primarily of bacterial DNA and provides instructions for maintaining the DNA insert prior to plant transformation. The skeletal part is not transfected into the plant cell. The various elements of the skeleton are listed in Table 1.

Example 2 Plant DNA insert and open reading frame (ORF)
The DNA insertion region used in pSIM1278 is 10,147 bp long (19,469 bp to 19,660 bp and 1 bp to 9,956 bp). The DNA insert consists only of native DNA and is stably integrated into the potato gene. The DNA insert and its functional part is only the genetic material of the vector pSIM1278 and is incorporated into the potato plant variety of the present invention. DNA inserts are listed in FIG. 2 and Table 2 below.

  The DNA inserts listed in Table 2 used to make the potato line E12 of the present invention do not activate the adjacent genes and do not adversely affect the phenotype of the potato plant variant E12. In addition, the potato plant variant E12 of the present invention does not produce a novel protein associated with the open reading frame encoded by the DNA insert.

Example 3 Agrobacterium strain and transfection The Agrobacterium strain AGL1 derived from C58 was isolated from the highly virulent plasmid pTiBo542 (Lazo et al., 1991). Developed by precise loss of transported DNA. Transposon insertion in the general recombinant gene (recA) stabilizes a recombinant plasmid vector such as pSIM1278 (FIG. 1). AGL1 exhibits resistance to carbenicillin and rifampicin and is removed from potato tissue transformed with timentin.

  Russett Burbank variety stock plants were maintained in a magenta box with 40 ml of 3% sucrose and 2 g / l gelrite containing half strength M516 medium (growth medium). A 4-6 mm potato internode segment was cut from a 4 week old plant, infected with Agrobacterium AGL1 strain carrying pSIM1278, and tissue culture medium containing 3% sucrose and 6 g / l agar (co-cultivation medium) Moved to. The infected explants were transferred to M404 medium containing 3% sucrose, 6 g / l agar and 150 mg / l timementin after 2 weeks to remove Agrobacterium (hormone-free medium). Details of this method can be found in Richard et al. (2008).

  One month later, the infected explants were transferred to fresh medium without any synthetic hormone and incubated at 24 ° C. in a Percival growth chamber under a 16 hour photoperiod. Here they began to form shoots. Many shoots expressed the ipt gene and showed a cytokinin overexpression phenotype; these shoots were not considered for further analysis. PCR genotyping demonstrated that about 0.3-1.5% of the remaining shoots contained at least a portion of P-DNA while lacking the ipt gene. Therefore, no marker was used to select transformed plants. The ipt-based marker-free plant traits are described in Richard et al. (2008).

  The process of removing Agrobacterium was started 2 days after explant infection. For this purpose, the tissue was exposed to the antibiotic timentin (150 mg / L) until it proved free of live Agrobacterium. Proof was obtained by incubating the stem fragment of the transformation event on a nutrient broth-yeast extract (NBY medium) at 28 ° C. for 2 weeks (repeated twice). According to 97 CFR part 340, transformed plants were transported and planted outdoors only in the absence of live Agrobacterium.

  Potato plant variant E12 was analyzed by DNA gel blot to determine the structure and copy number of the integrated DNA insert and to confirm the absence of vector backbone sequences. In addition, molecular characterization was used to sequence the junction adjacent to the DNA insert to indicate the stability of the inserted DNA. The junction sequencing information provided the basis for developing a specific PCR test for the in-gene potato plant variant E12. Potato cultivar E12 was derived from hybridization results where various DNA digests hybridized to AGP, ASN, PHL and GBS molecular probes and were found to contain one complete copy of the DNA insert.

Example 4 Evidence for the absence of vector backbone DNA Unlike many commercially available transgenic crops, the potato cultivar E12 of the present invention is free of Agrobacterium-derived sequences used for transformation of vector backbone DNA and the like. This has been confirmed by three different methods: 1) First, the presence or absence of a negative selectable isopentenyl isomerase (ipt) marker gene in the vector backbone, the ipt gene expression and the resulting cytokinin type Determined as accidental transport of skeletal DNA containing the Aptacterial-derived ipt gene expression cassette, which causes formation of the hormone isopentenyl adenosine, 2) Southern blot hybridization was then performed on transformed potato plants. Used and skeleton DN A first screening method to confirm the absence of A, and 3) Amplification of a fragment indicating a region in the backbone DNA adjacent to the junction between the border region and the adjacent backbone DNA or the DNA insert A PCR was designed to do this. The effectiveness of the method was confirmed using pSIM1278 DNA as a positive control. The potato cultivar E12 of the present invention did not produce a PCR band that is an indicator of the presence of vector backbone DNA.

Example 5 FIG. Stability of the inserted DNA The stability of the DNA insert was evaluated using DNA gel blot hybridization and trait evaluation, both in the original transformants and again in the grown plant material. These studies were conducted to confirm that intragenic events expressed a consistently integrated trait in a reliable manner. Instability can be caused by rare recombination events or can also be caused by methylation. Since potatoes are usually propagated clonally, standard assessments for reproductive crops are not directly applicable, and tubers rather than seeds were used to determine subsequent generations. DNA blot hybridization results showed that a certain band was present in many generations and was therefore stable. Further evidence for stability was obtained by confirming trait efficacy in 1 and 2 tuber seed generations.

  DNA insert stability was demonstrated by extracting and evaluating DNA in leaves of plants that were grown in vitro and not planted in soil in the original transformant (G0). For first generation (G1) analysis, two growing plants from each intragenic variant and one plant from each control were planted in the greenhouse; one of the tubers harvested from each plant was transferred to the G1 plant. Planted to obtain leaves, which were used to isolate DNA and evaluate the G1 generation. Tubers from this generation were replanted and the properties of this generation were characterized by the leaves of the resulting G2 plants.

  In all lanes, hybridization with GBS probe of DNA isolated from Russet Burbank potato cultivar E12 of the present invention revealed two common bands (7.2 and 8.0-kb), and AGP Hybridization with the probe revealed three bands (1.4, 4.2 and 4.9-kb). These bands are indicative of the DNA fragment of the unmodified genome. Presence of two additional bands in DNA isolated from all intragenic material (the 2.2-kb band is indicative of the internal DNA insert, while the other (4.4-kb for E12 and GBS, 2.15-kb) for E12 and AGP represent DNA insert junction fragments), where the insert of the original transformant (G0) is stable in the first and second asexual generations G1 and G2. Indicates that it is maintained.

  A catechol assay for Ppo activity provided further evidence for DNA insert stability in the E12 variant. All E12 tubers remained uncolored (FIG. 3), while non-transformed material was stained. This demonstrated that variant E12 has the ability to silence the Ppo5 gene in G2 tubers after two years of field trials. The preparation of field test materials included repeated tissue culture and asexual reproduction without affecting the stability of the inserted genetic material.

Example 6 Junction analysis and variant-specific detection At least one DNA insert / adjacent DNA junction was sequenced using either adapter-ligation mediated PCR or thermal asymmetric interlaced PCR. The junction sequence was used to design primers for potato cultivar E12, and these primers were applied for the variant-specific PCR-based detection method. This developed method was used to monitor plants and tubers in the field and in storage to confirm the absence of intragenic material in tubers or processed foods and to ensure the purity of organic seeds.

Example 7 Efficacy and tissue specificity of gene silencing Using gene silencing methods, reducing the activity of native proteins of Asn1, Ppo5, PhL and R1, assessing transcript levels rather than protein levels, and novel expression Linked type traits and changes at the molecular level.

  Strong silencing of the Asn1 gene involved in ASN (asparagine) formation in leaves and stems can adversely affect growth, so it is specific for tubers and running roots and is less active in photosynthetic active tissues and roots The Agp promoter and the Gbss promoter were used to drive gene silencing in tubers and running roots. Transcript levels of four target genes in various tissues of plant variant E12 and untransformed control were determined by Northern blot analysis.

  In non-transformed control tubers, transcript levels are “high” for the Asn1, PhL and R1 genes (easily detectable by Northern blot hybridization). “Low” for the Ppo5 gene. Northern blot comparisons showed that Asn1, Ppo5 and PhL were similarly expressed in tubers in the greenhouse and in the field. In contrast, the R1 gene was slightly more effectively silenced in the greenhouse grown control tubers than the tubers from the field.

  In the varieties E12 tubers, strongly reduced transcript levels for the Asn1 and Ppo5 genes were associated with low acrylamide latency and reduced discoloration associated with black spot.

  The transcript level for the PhL gene was partially reduced in E12 tubers. This change was associated with a reduction in the amount of glucose and fructose. The R1 transcript was partially reduced (“whipped”) in E12 tubers, helping to limit the degradation of starch to sugar.

  Asn1, Ppo5 and R1 genes were expressed at low levels in the roots of non-transformed plants. In contrast, high transcript levels in the control were associated with the PhL gene.

  Transcript levels of Asn1 and Ppo5 genes were still lower in the Russet Burbank E12 plants grown in the greenhouse than in the control roots. This modification was expected and was not associated with any unwanted or unusual new phenotype. PhL gene expression was also down-regulated in the root of intragenic variant E12.

  The amount of transcript for the R1 gene was slightly reduced ("whispered") in the root of variant E12. This molecular change contributed to limited degradation of starch to sugar.

  In leaf tissue, transcript levels for the Asn1 gene were similar for E12 lines and untransformed counterparts. Transcript levels for Ppo5 gene expression were undetectable in all cases, while levels for the PhL gene were consistently higher between the original Russett Burbank variant and the transformed derivative E12. The transcript level for the R1 gene was not changed in variant E12 when compared to its subject.

  In stem tissue, Asn1 gene transcript levels were similar for event E12 and its subjects. Transcript levels for the Ppo5 gene were reduced in variant E12 of the present invention. PhL gene expression was very similar to the E12 line and its controls, and R1 gene expression was not reduced.

  In root tissue, transcript levels for both Asn1 and Ppo5 genes were reduced in variant E12. These results indicated that the promoter used to drive silencing was partially functional in underground tissue.

  In floral tissue, Asn1 gene transcript levels were lower in the variant E12 of the present invention than in the original variant Russet Burbank. Transcripts were not detectable for the Ppo5 gene and the expression levels of the PhL and R1 genes were similar to the control.

  These results indicate that the expression levels of Asn1 and Ppo5 genes are down-regulated in tubers and roots in potato variety E12, and that R1 and PhL genes are partially silenced in tubers and roots in variant E12. It was proved to be singed. Silencing was most effective on tubers and roots.

  The selected potato variant E12 had a stronger effect on the expression levels of the Asn1 and Ppo5 genes than on the expression levels of the R1 and PhL genes. These results show that the inventor's objectives (1) prevent the formation of Ppo protein and free ASN as cooperatively as possible, and (2) the conversion of starch to glucose and fructose, in part Only matched with blocking.

  Time-dependent changes in transcript levels in tissues other than tubers and running roots demonstrated some leakage of tuber / running root promoters. In addition, small RNAs produced in tubers through expression of silencing cassettes can migrate to other tissues, particularly roots and stems (Molnar et al., 2010). In most cases of changes in expression levels in tissues other than tubers and running roots, this difference was slight. A summary of down-regulated transcript levels in specific tissues of potato cultivar E12 is shown in Table 3. In Table 3, A = Asn1, P = Ppo5, L = PhL and R = R1. The underlined letters in Table 3 indicate a strongly downregulated gene expression level (less than one half), otherwise the expression level is downregulated without less than one half. The

Example 8 FIG. Field Performance and Tuber Evaluation 2009, 2010 and 2011 tests were mechanically planted to facilitate harvesting to ensure that the in-gene potato was separate from the unmodified material. For the 2009 assessment, “nuclear seed” microtubers from each event and control varieties were used to plant 4-5 plots in a row (20 microtubers per plot) and the plots were blocked across the field. Placed randomly within. This random complete block design (RCB) was typically for evaluation of new potato variants and events. The approach taken in 2010 and 2011 used one random event per location and two random plots per control, and the same number of blocks or the same number of iterations (number of plots per event): An RCB design was also used. Each plot in 2010 consists of three rows of 20 seed pieces from the first generation (G1) tubers produced at Cherry County, NE for Russet Burbank in 2009. The Russet Burbank seed for the 2011 test was a second generation (G2) produced in 2010 at Cherry County, NE. In all tests, seeds of intragenic lines were manipulated in the same manner as their unmodified controls. Tubers grown outdoors are more desirable than small tubers as seeds. This is because they produce uniform plants that grow better and produce higher yield and quality tubers.

  Each plot, in some cases, using a normalized monitoring scale for differential responses to insect, disease and environmental stresses that are not artificially induced but occur spontaneously during the growing season, Qualitatively evaluated. Mid-season monitoring was conducted in 2009, 2010 and 2011 immediately before or during the initial row closure and flowering (June-July in most tests except the Florida test evaluated in April) Strength, leaf color, leaf size, leaf winding, disease symptoms (presence / absence) and insect-related plant damage were assessed. In 2011, specific insect, disease and abiotic stress factors common to the growing area were evaluated. Disease and insect pressure are generally highest in the middle and late seasons, but plants were monitored from July to September for symptoms caused by pathogens and insects (March to May for the Florida test). Late monitoring of vine maturity and disease was performed once prior to vine killing with the intention of ensuring tuber maturation and late skin formation. Vine killing is done either by vine harvesting or hammering, or by using an approved herbicide such as Regrone according to the manufacturer's recommendations (JR Johnson, Roseville, MN). At this point, the plants were also evaluated for disease symptoms and insect damage. In some cases where disease symptoms were identified, sprouting tests were also conducted to confirm the trial.

Mean values, standard deviations and 90% confidence intervals were calculated using JMP 9.0.2. Conventional variant ranges were created by obtaining the minimum and maximum average values (year ^* location ^* entries) of all conventional variants and including them in the experiment. All features for Russett varieties were analyzed in JMP 9.0.2 by combining data from multiple years and multiple locations.

Example 9 Summary of potato cultivar E12 characterization potato cultivar E12 is due to reducing acrylamide through reducing the expression of enzymes responsible for black spot and reducing the concentration of reactants, ie asparagine and reducing sugars. Oriented, quality improvement. Potato cultivar E12 was transformed with a nucleic acid sequence that was native to the potato plant genome and did not contain foreign DNA, Agrobacterium DNA, viral markers or vector backbone sequences. In addition, we conducted an agricultural study and obtained events that grew up as in the conventional control except for the features associated with this trait.

Agricultural Characteristics Tables 4-7 show the evaluation of potato cultivar E12 events grown in 2009, 2010 and 2011 and the agricultural characteristics of the controls. Results were analyzed by statistical methods when possible. The overall data showed no major differences between the Russset Burbank control and the E12 Russset Burbank event.

  Table 4 shows the number of site years for each feature tested for variant E12. The agronomic characteristics for E12 and Burbank controls are shown in Table 5. For 7 agricultural features, no statistically significant difference was detected between E12 and the control. The vine maturity score data could not be compared statistically and the value of E12 was outside the range of the combined conventional variants (3.65 vs. 3.00 to 3.50, respectively) . For plant forces, statistically significant differences were detected between E12 and controls (3.38 vs. 3.08), but E12 values were within the conventional variety range. In Table 5, stem data per plant is only from 2011; conventional varieties (ConV) were equal to the average range of conventional Ranger, Burbank and Atlantic varieties; NA was statistical Indicates that no pharmacological comparison was possible.

  Yields and grades for E12 and Burbank controls are shown in Table 6. No statistically significant differences were detected for total yield, specific gravity, high sugar or sugar terminus. Statistical analysis was not possible for the entire internal defect, but the value for E12 was within the range of conventional variants. Four differences were detected between E12 and the control: 4-6 oz. Category (25.3 vs 21.5), 6-10 oz. Category (33.1 vs 30.3), 10-14 oz. Category (12.6 vs 16.2), and 14 oz. More than 8.9 categories (8.9 vs 13.1). All values for E12 for these differences were within conventional varieties. In Table 6, specific gravity data is only from 2011; the conventional variant (ConV) was equal to the average range of the conventional Ranger and Burbank variants; NA was not statistically comparable Indicates that it was possible.

  The color of the E12 and Burbank control flowers are shown in Table 7. Both purple / mixed color flowers and white flowers were observed in different plots for each entry.

  Based on the data presented in Tables 4-7 for potato cultivar E12, agricultural characteristics, flower color, yield and grade, and economic interaction between untransformed Russset Burbank varieties and potato cultivar E12 We concluded that there was no major difference in action. Therefore, based on multi-year data, the Ranger Russet variant E12 raised that there is no significant risk of persistence in the environment as a result of possible weed or pest damage.

Black spot resistance Black spot damage is the discoloration of damaged tubers and represents one of the most important quality problems in the potato industry. This condition is the result of leakage of polyphenol oxidase (Ppo) from the damaged plastid into the cytoplasm. The enzyme oxidase then forms a dark precipitate in the cytoplasm. One of the two silencing cassettes of the pSIM1278 DNA insert contains two copies of the Ppo5 gene fragment from Solanum velcosum as inverted repeats between regulatory elements. Expression of this inverted repeat causes silencing of the potato Ppo5 gene and significantly reduces the appearance of black spot.

  Tubers grown in the field of potato cultivar E12 and a control Russet Burbank variant sensitive to black spots were assayed for black spot resistance in two ways.

  Tuber discoloration is caused by the activity of polyphenol oxidase, which oxidizes phenols containing catechol and produces compounds that rapidly polymerize to produce pigments. An indirect method of testing for black spot resistance is to pipette 1 ml of catechol (25 mM, 50 mM MOPS, pH 6.5) onto the cut surface of the tuber and monitor the Ppo-dependent development of dark brown precipitates. It is to be. Ppo-dependent development of dark brown precipitate was assessed after 20 minutes. Russet Burbank control and Variety E12 tubers were harvested from three different plants grown in 2009 in Canyon County, ID. The results of the catechol assay for potato cultivar E12 are shown in FIG. As shown in FIG. 3, the Russet Burbank control turned dark brown and showed black spots. On the other hand, potato cultivar E12 did not turn dark brown, indicating that E12 is more resistant to black spots than the non-transformed Russet Burbank control.

  A second method for assaying for wound resistance is to rotate nine tubers of control and potato cultivar E12, each harvested from Canyon County, ID in 2009, to 10 revolutions in a wound barrel (rotary drum), at room temperature. Incubated for 3 days, and then mechanically peeled to count the number of both black and line cracked (cracked) plaques. Table 8 shows the results expressed as the average value of hair growth of three experiments, and the error bar indicates the standard error of the average value. Significance was determined using Student's two-tailed t test (p <0.05).

  As shown in Table 8, the barrel wound test showed undetectable black spot levels in potato E12 of the present invention. These results show that the varieties E12 tubers are more resistant to black spots than the non-transformed control Russet Burbank variety tubers, and that the pSIM1278 DNA insert is impact-induced with black spots. Indicates to cause discoloration.

Asparagine and acrylamide levels Silencing of the asparagine synthetase gene resulted in an average reduction of 74% of free asparagine in potato cultivar E12. The lower level of asparagine that combines with reducing sugars in the Maillard reaction to form acrylamide results in a 67% reduction of acrylamide in the fried food. The differences in asparagine and acrylamide between the Russet Burbank control and potato E12 are shown in Table 9. Prior to testing for acrylamide, the control and E12 were French fries. All results were from tubers analyzed at a time close to harvest.

  Potato cultivar E12 is an improved quality Burset variety with reduced expression of enzymes responsible for black spots and reduced acrylamide levels.

Further embodiments of the invention The work leading to potato varieties that combine the advantageous features described above is largely empirical. This study requires significant research time, effort and expense. The development of potato cultivars often spends more than 8 years from the greenhouse to commercial use. Breeding begins with the careful selection of a good parent to incorporate the most important features into the offspring. Since all desired traits usually do not appear by a single cross, reproduction will always be cumulative.

  This breeding technique is followed by controlled pollination of the parent clone. Typically, pollen is collected in gelatin capsules for later use when pollinating female parents. Hybrid seeds are planted in a greenhouse and tubers are harvested and retained from thousands of individual seedlings. The next year, 1 to 4 tubers from each obtained seedling are planted outdoors, where great care is taken not to disperse viruses and diseases. From this first year seedling harvest, several “seed” tubers from each hybrid solid that survived the selection process are retained for planting the next year. After the second year, samples are taken for density measurement and fried test to determine the suitability of the tubers for commercial use. Plants that survived the selection process up to this point are then planted in a larger capacity in the third year for a more comprehensive series of fried food tests and density determination. In the 4th year of the development phase, the survivors of the selection are subjected to field trials under several conditions to determine their suitability for different growth conditions. Eventually, varieties with excellent quality will be transferred to other farms and seeds will be increased to market scale. In general, by this point, it is contemplated to investigate cropping, harvesting and testing over 8 years to develop new and improved potato cultivars.

  With the advent of molecular biology techniques that have allowed the isolation and characterization of genes that code for specific protein products, scientists in the field of plant biology have expressed the plant genome, including foreign genes. In addition, there has been a strong interest in manipulating expression to include additional or modified forms (possibly driven by different promoters) of native or endogenous genes. Such exogenous additional genes are collectively referred to herein as “transgenes”. Over the last 15-20 years, several methods for producing transgenic plants have been developed, and the present invention also relates to the claimed varieties or lines of transformed varieties in a detailed embodiment.

  Plant transformation involves the construction of an expression vector that functions in plant cells. Such vectors include DNA comprising a gene under the control of a regulatory sequence (eg, a promoter) or operably linked to a regulatory sequence. An expression vector can include one or more such operably linked gene / regulatory element combinations. The vector (s) may be in the form of a plasmid, may be used alone or in combination with other plasmids, and may be used to isolate a transgene, as described below. A transformed potato plant is provided using a transformation method that is incorporated into the genetic material of the plant.

  Conventional plant propagation typically relies on random recombination of plant chromosomes to produce varieties with new and improved characteristics. In accordance with standard well-known techniques, a gene “expression cassette” containing genes and regulatory elements is inserted within the boundaries of Agrobacterium-isolated delivery DNA (“T-DNA”) and incorporated into the plant genome. Agrobacterium-mediated transfer of T-DNA material typically involves the following standard procedures: (1) For selection of transformation by in vitro recombination of genetic elements of at least one exogenous source (2) This expression cassette is usually hundreds of bases of Agrobacterium DNA flanked by T-DNA border sequences, often together with at least one other expression cassette containing exogenous DNA Inserting into the T-DNA region of a binary vector of pairs, (3) sequences located between T-DNA boundaries often with some or all of the additional binary vector sequences from Agrobacterium Together, incorporating into plant cells, and (4) selecting stably transformed plant cells that exhibit the desired trait. This trait is, for example, increased yield, improved power, increased resistance to disease and insects, or increased ability to survive under stress.

  Thus, the genetic engineering method involves the identification and selection of exogenous, resident, regulatory elements such as promoters and terminators and the expression of new traits or transformants from viruses, bacteria and plants. Rely on nucleic acids, including genes involved in functioning as markers for Marker genes are typically derived from bacterial sources and confer antibiotic resistance or herbicide resistance. Classical breeding methods are laborious and time consuming, and new varieties typically show only one relatively mild improvement.

  In “antisense” technology, the sequence of the native gene is reversed to silence the expression of the gene in the transgenic plant. However, reverse DNA usually contains a new and uncharacterized open reading frame inserted between the promoter and terminator, which encodes an exogenous amino acid sequence and interferes with plant growth, And / or may reduce nutritional value and may be undesirable.

Expression vector for potato transformation: Marker gene The expression vector comprises at least one genetic marker operably linked to a regulatory element (eg a promoter), which contains a negative selection, ie a selective marker gene Either by inhibition of cell growth or by positive selection, ie screening for the product encoded by the genetic marker. A number of selectable marker genes commonly used for plant transformation are well known in the transformation field, such as enzymes that metabolically detoxify selective chemical agents such as antibiotics or herbicides. A gene that encodes or a modified target that is insensitive to inhibitors. Several positive selection methods are also known in the art.

  One commonly used selectable marker for plant transformation is the neomycin phosphotransferase II (nptII) gene, which confers resistance to kanamycin when under the control of plant regulatory signals. Fraley et al. , Proc. Natl. Acad. Sci. U. S. A. , 80: 4803 (1983). Another commonly used selectable marker gene is the hygromycin transferase gene, which confers resistance to the antibiotic hygromycin. Vanden Elzen et al. , Plant Mol. Biol. 5: 299 (1985).

  Additional selectable marker genes are genes of bacterial origin that confer resistance to antibiotics, including gentamicin acetyltransferase, streptomycin phosphotransferase, and aminoglycoside-3'-adenyltransferase, which is a bleomycin resistance determinant. Hayford et al. , Plant Physiol. 86: 1216 (1988), Jones et al. Mol. Gen. Genet. 210: 86 (1987), Svab et al. , Plant Mol. Biol. 14: 197 (1990) Hille et al. , Plant Mol. Biol. 7: 171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil. Comai et al. , Nature 317: 741-744 (1985), Gordon-Kamm et al. , Plant Cell 2: 603-618 (1990) and Stalker et al. , Science 242: 419-423 (1988).

  Selectable marker genes for plant transformation that are not of bacterial origin include, for example, mouse dihydrofolate dehydrogenase, plant 5-enolpyruvirshikimate-3-phosphate synthase, and plant acetolactate synthase. Eichholtz et al. , Somatic Cell Mol. Genet. 13:67 (1987), Shah et al. , Science 233: 478 (1986), Charest et al. , Plant Cell Rep. 8: 643 (1990).

  Another class of marker genes for plant transformation requires screening of putative transformed plant cells rather than direct gene selection of transformed cells for resistance to toxic substances such as antibiotics. These genes are particularly useful in quantifying gene expression in specific tissues or visualizing spatial patterns and are called reporter genes. This is because they can be fused to genes or gene regulatory sequences for investigation of gene expression. Commonly used genes for screening putative transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase and chloramphenicol acetyltransferase. Jefferson, R.A. A. , Plant Mol. Biol. Rep. 5: 387 (1987), Teeri et al. , EMBO J. et al. 8: 343 (1989), Koncz et al. , Proc. Natl. Acad. Sci. USA 84: 131 (1987), DeBlock et al. , EMBO J. et al. 3: 1681 (1984). .

  In vivo methods for visualizing GUS activity that do not require the destruction of plant tissue are available. Molecular Probes publication 2908, IMAGEGEN GREEN, p. 1-4 (1993) and Naleway et al. , J .; Cell Biol. 115: 151a (1991). However, these in vivo methods for visualizing GUS activity should be useful for recovering transformed cells due to low sensitivity, high fluorescence background, and limitations associated with the use of luciferase as a selectable marker. Has not been proven.

  More recently, a gene encoding green fluorescent protein (GFP) has been used as a marker for gene expression in prokaryotic and eukaryotic cells. Chalfie et al. , Science 263: 802 (1994). GFP and mutated ties of GFP can be screenable markers.

Expression Vector for Potato Transformation: Promoter Genes that can be included in the expression vector must be driven by a nucleotide sequence that contains regulatory elements, such as a promoter. Several types of promoters are well known in the transformation art, as are other regulatory elements that can be used alone or in combination with a promoter.

  As used herein, “promoter” includes a reference to a region of DNA upstream from the start of transcription that is involved in the recognition and binding of RNA polymerase and other proteins that initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in specific tissues such as leaves, roots, seeds, fibers, wood conduits, temporary conduits or thick walls. Such a promoter is referred to as “tissue suitable”. Promoters that initiate transcription only in specific tissues are termed “tissue specific”. “Cell type” specific promoters drive expression primarily in specific cell types of one or more organs, for example, vascular cells in the roots or leaves. An “inducible” promoter is a promoter that is under environmental control. Examples of environmental conditions that can affect transcription by an inducible promoter are anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter that is active under most environmental conditions.

A. Inducible promoters Inducible promoters are operably linked to genes for expression in potato. Optionally, the inducible promoter is operably linked to a nucleotide sequence that encodes a signal sequence that is operably linked to a gene for expression in potato. Inducible promoters increase the rate of transcription in response to an inducing agent.

  Any inducible promoter can be used in the present invention. Ward et al. , Plant Mol. Biol. 22: 361-366 (1993). Examples of inducible promoters are those derived from the ACEI system in response to Exemplary inducible promoters include, but are not limited to, that from the copper (Mett et al., PNAS 90: 4567-4571 (1993); benzene). In2 gene from maize (Hershey et al., Mol. Gen Genetics 227: 229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243: 32-38 (1994) in response to amide herbicide toxicity reducers. )), Or Tn10-derived Tet repressor (Gatz et al., Mol. Gen. Genetics 227: 229-237). 1991)), and the like. Particularly preferred inducible promoters are those that respond to inducing agents that are not normally responsive to plants. An exemplary inducible promoter is an inducible promoter from a steroid hormone gene, whose transcriptional activity is induced by glucocorticosteroid hormones. Schena et al. , Proc. Natl. Acad. Sci. USA 88: 0421 (1991).

B. A constitutive promoter is a nucleotide that encodes a signal sequence that is operably linked to a gene for expression in potato, or a constitutive promoter is operably linked to a gene for expression in potato Operatively linked to the array.

  Many different constitutive promoters can be utilized in the present invention. Exemplary constitutive promoters include plant virus-derived promoters such as the CaMV-derived 35S promoter (Odell et al., Nature 313: 810-812 (1985)) and rice actin (McElroy et al., Plant Cell 2: 163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12: 619-632 (1989) and Christensen et al., Plant Mol. Biol. 18: 675-689 (1992)); (Last et al., Theor. Appl. Genet. 81: 581-588 (1991)); MAS (Velten et al., EMBO J. 3: 2723- 730 (1984)) and maize H3 histones (Lepetit et al., Mol. Gen. Genetics 231: 276-285 (1992) and Atanasova et al., Plant Journal 2 (3): 291-300 (1992)). It is a promoter derived from a gene.

  The ALS promoter, Xba1 / Ncol fragment (or a nucleotide sequence similar to this Xba1 / Ncol fragment) that is 5 'to the ALS3 structural gene represents a particularly useful constitutive promoter. See PCT application WO 96/30530.

C. Tissue-specific or tissue-preferred promoter A tissue-specific promoter is operably linked to a gene for expression in potato. Optionally, the tissue specific promoter is operably linked to a nucleotide sequence that encodes a signal sequence that is operably linked to a gene for expression in potato. Plants transformed with the gene of interest operably linked to a tissue-specific promoter produce the protein product of the transgene exclusively or preferentially in the specific tissue.

  Either tissue specific or tissue preferred promoters can be utilized in the present invention. Exemplary tissue specific or tissue preferred promoters include root preferred promoters such as those derived from the phaseolin gene (Murai et al., Science 23: 476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad.Sci.USA 82: 3320-3324 (1985)); leaf-specific and light-inducible promoters such as those derived from cab or rubisco (Simpson et al., EMBO J.4 (11): 2723-2729 ( 1985) and Timko et al., Nature 318: 579-582 (1985)); sputum specific promoters such as those derived from LAT52 (Twell et al., Mol. Gen. Genetics). 217: 240-245 (1989)); pollen-specific promoters such as those derived from Zm13 (Guerrero et al., Mol. Gen. Genetics 244: 161-168 (1993)) or small such as those derived from apg Spore suitable promoters (Twell et al., Sex. Plant Reprod. 6: 217-224 (1993)) can be mentioned.

Signal sequence to target the protein to the intracellular compartment Transport of the protein produced by the transgene to an intracellular compartment such as chloroplast, vacuole, peroxisome, glyoxisome, cell wall or mitochondria, or to apoplast Transport for secretion is achieved by operable linkage of a nucleotide sequence encoding a signal sequence to the 5 ′ and / or 3 ′ region of the gene encoding the protein of interest. Targeting sequences at the 5 ′ and / or 3 ′ ends of structural genes can determine where the encoded protein is ultimately compartmentalized during protein synthesis and processing.

  The presence of the signal sequence directs the polypeptide for secretion into an organelle or subcellular compartment, or apoplast. Many signal sequences are known in the art. For example, Becker et al. , Plant Mol. Biol. 20:49 (1992); Close, P.M. S. , Master's Thesis, Iowa State University (1993); Knox, C .; , Et al. , Plant Mol. Biol. 9: 3-17 (1987); Lerner et al. , Plant Physiol. 91: 124-129 (1989); Frontes et al. Plant Cell 3: 483-496 (1991); Matsuoka et al. , Proc. Natl. Acad. Sci. 88: 834 (1991); Gould et al. , J .; Cell. Biol. 108: 1657 (1989); Creissen et al. Plant J .; 2: 129 (1991); Kalderon, et al. , Cell 39: 499-509 (1984); Steifel, et al. , Plant Cell 2: 785-793 (1990).

Exogenous protein genes and agricultural genes Exogenous proteins can be produced in commercial quality by the transgenic plants according to the present invention. Thus, techniques well known in the art for selection and propagation of transformed plants yield a plurality of transgenic plants that are harvested in a conventional manner and then the exogenous protein is then transferred to the tissue or whole of interest. Extracted from the biomass. Protein extraction from plant biomass can be accomplished, for example, by known methods discussed by: Henry and Orr, Anal. Biochem. 114: 92-6 (1981).

  According to a preferred embodiment, the transgenic plant produced as a foreign protein market product is a potato plant. In another preferred embodiment, the target biomass is seeds or tubers. For a relatively small number of transgenic plants exhibiting high levels of expression, a genetic map can first be generated via conventional RFLP, PCR and SSR analysis. Our analysis identifies the approximate chromosomal location of the integrated DNA molecule. For an exemplary methodology in this regard, see Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology CRC Press, Boca Raton 269: 284 (1993). Map information regarding the chromosomal location is useful for patent protection of the present transgenic plant. If unauthorized growth occurs and mating with other germplasm is made, the map of the integration region can be compared to a similar map for the suspicious plant, with the latter sharing the percentage with this plant Determine whether or not. Map comparison may include hybridization, RFLP, PCR, SSR and sequencing, which are conventional techniques.

  Similarly, agricultural genes can be expressed in transformed plants using the present invention. More particularly, plants can be genetically engineered to express various phenotypes for agricultural purposes. Exemplary genes related to this point include, but are not limited to, those in the categories shown.

1. Genes conferring resistance to pests and diseases and these encode:
A. Plant disease resistance gene plant defense is often activated by specific interactions between the product of the disease resistance gene (R) and the product of the corresponding non-pathogenic (Avr) gene in the pathogen. . Plant varieties can be transformed with cloned resistance genes to engineer plants to be resistant to specific pathogen strains. For example, Jones et al. , Science 266: 789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporum fulvum); Martin et al. , Science 262: 1432 (1993) (tomato Ptogene for resistance to Pseudomonas syringae pv. Tomato encoders a protein kinase); Mindrinos et al. Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance to pseudomonas syringae).
B. See genes conferring resistance to pests such as soybean cyst nematode, for example PCT application WO 96/30517; PCT application WO 93/19181.
C. Bacillus thuringiensis protein, derivatives thereof, or synthetic polynucleotides modeled thereon, for example, the cloning and nucleotide sequence of the Bt δ-endotoxin gene, Geiser et al. Gene 48: 109 (1986). In addition, DNA molecules encoding the δ-endotoxin gene are commercially available from the American Type Culture Collection, Manassas, Virginia, for example under ATCC accession numbers 40098, 67136, 31995 and 31998.
D. Lecithin, for example, Van Damme et al. Plant Molec. Biol. 24:25 (1994). This discloses the nucleotide sequence of several Clivia miniata mannose-binding lecithin genes.
E. See PCT application US 93/06487, a vitamin binding protein such as avidin. This teaches the use of avidin and avidin homologs as larvicides against insect pests.
F. Enzyme inhibitors such as protease inhibitors or proteinase inhibitors or amylase inhibitors. For example, Abe et al. , J .; Biol. Chem. 262: 16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huub et al. Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani et al. Biosci. Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus α-amylase inhibitor) and US Pat. No. 5,494,813 (Hepher and Atkinson, issued February 27, 1996).
G. Insect-specific hormones such as ecdysteroids or juvenile hormones, variants thereof, mimetics based on them, or antagonists or agonists thereof, for example, Hammock et al. , Nature 344: 458 (1990), see the disclosure of baculovirus expression of cloned juvenile hormone esterase (juvenile hormone inactivator).
H. Insect-specific or neuropeptides that disrupt the physiology of the pest affected during expression, such as Regan, J. et al. Biol. Chem. 269: 9 (1994) (expression cloning yields DNA encoding an insect diuretic hormone receptor), and Pratt et al. Biochem. Biophys. Res. Comm. 163: 1243 (1989) (allostatin is identified in Diploptera puntata). See also US Pat. No. 5,266,317 to Tomalski et al. This discloses a gene encoding an insect-specific paralytic neurotoxin.
I. An insect-specific poison produced in nature by snakes, wasps, etc. For example, Pang et al. , Gene 116: 165 (1992) for disclosure of heterologous expression in plants of genes encoding scorpion insecticidal peptides.
J. et al. Enzymatic response to excessive accumulation of monoterpenes, sesquiterpenes, steroids, hydroxamic acids, phenylpropanoid derivatives or other non-protein molecules with insecticidal activity.
K. Enzymes involved in modifications, including post-translational modifications of biologically active branches, such as glycolytic enzymes, proteolytic enzymes, lipolytic enzymes, nucleases, cyclases, transaminases, esterases, hydrolases, phosphatases, kinases, phosphorylases, polymerases, Elastase, chitinase and glucanase, either natural or synthetic. See PCT application WO 93/02197 (Scott et al.). This discloses the nucleotide sequence of the calase gene. A DNA molecule comprising a chitinase coding sequence can be obtained, for example, from ATCC under accession numbers 39637 and 67152. Also, Kramer et al. , Insect Biochem. Molec. Biol. See also 23: 691 (1993). This teaches a nucleotide sequence encoding tobacco immature chitinase. And Kawalleck et al. Plant Molec. Biol. 21: 673 (1993). This provides the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.
L. Molecules that stimulate signal transduction, for example, Botella et al. Plant Molec. Biol. 24: 757 (1994), disclosure of the nucleotide sequence of mungbean calmodulin cDNA clone, and Griess et al. , Plant Physiol. 104: 1467 (1994) (providing the nucleotide sequence of the soybean calmogillin cDNA clone).
M.M. Hydrophobic Moment Peptide See: PCT application WO 95/16776, which discloses peptide derivatives of tachypressin that inhibit fungal plant pathogens. As well, PCT application WO 95/18855 teaches synthetic antimicrobial peptides that confer disease resistance.
N. A membrane permease, channel former or channel blocker. For example, Jaynes et al. , Plant Sci 89:43 (1993), see the disclosure of heterologous expression of cecropin-β lytic peptides conferring resistance to Pseudomonas solanacearum in transgenic tobacco plants.
O. Accumulation of viral invasive proteins or complex toxins derived therefrom, such as viral coat proteins in transformed plant cells confers resistance to infection and / or the onset of such diseases by viruses from which the encoded protein is derived and / or related viruses . Beachy et al. , Ann. Rev. Phytopathol. 28: 451 (1990). Coat protein-mediated resistance has been conferred on transformed plants against alfalfa mosaic virus, cucumber mosaic virus and tobacco mosaic virus.
P. Insect-specific antibodies or immunotoxins derived from them Thus, antibodies targeted against important metabolic functions in the insect gut inactivate the affected enzymes and kill the insects. Taylor et al. , Abstract # 497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzyme inactivation through production of single chain antibody fragments in transgenic tobacco).
Q. Virus specific antibodies such as Tavladoraki et al. , Nature 366: 469 (1993). This indicates that transgenic plants expressing the recombinant antibody gene are protected from viral attack.
R. Therefore, the fungal endo-α-1,4-D-polygalacturonase is naturally produced by the plant cell wall soluble homo-α-1,4-D-galacturonase Facilitates colonization and plant nutrient release. Lamb et al. Bio / Technology 10: 1436 (1992). Cloning and characterization of the gene encoding bean endopolygalacturonase inhibitor protein is described by Toubart et al. Plant J .; 2: 367 (1992).
S. Developmental stop proteins produced naturally by plants, such as Logemann et al. Bio / Technology 10: 305 (1992) show that transgenic plants expressing barley ribosome inactivation gene have increased resistance to fungal disease.
T.A. Genes involved in systemic acquired resistance (SAR) response and / or pathogenesis-related genes Briggs, S. et al. Current Biology, 5 (2) (1995).
U. Antifungal gene Cornelissen and Melchers, Plant Physiol. 101: 709-712 (1993); Parijs et al. , Planta 183: 258-264 (1991) and Bushnell et al. , Can. J. et al. of Plant Path. 20 (2): 137-149 (1998).
V. Genes conferring resistance to Phytophthora bright, for example, R1, R2, R3, R4 and other resistance genes Naess, S., et al. K. , Et. al. (2000) Resistance to late blight in Solanum bulbocastanum is mapped to chromosome Theor. Appl. Genet. 101: 697-704 and Li, X. , Et. al. (1998) Autotetraploids and genetic mapping using common AFLP markers: the R2 Theor. Appl. Genet. 96: 1121-1128.

2. A gene that develops tolerance to herbicides. For example:
A. Herbicides that inhibit growth points or meristems, such as imidazolinones or sulfonylureas Examples of genes in this category, for example, encode mutant ALS and AHAS enzymes, respectively, as described below: Lee et al . , EMBO J. et al. 7: 1241 (1988) and Miki et al. , Theor. Appl. Genet. 80: 449 (1990).
B. Glyphosate (reduced resistance by mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) and aroA genes, respectively) and glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus ( Streptomyces hygroscopicus (PAT bar gene) and other phosphono compounds such as pyridineoxy or phenoxypropionic acid and cyclohexane (ACCase inhibitor coding gene) such as Shah, et al, which discloses nucleotide sequences in the form of EPSP that confer glyphosate resistance . U.S. Pat. No. 4,940,835. The DNA molecule encoding the aroA gene is obtained under ATCC accession number 39256, and the nucleotide sequence of the mutant gene is disclosed in US Pat. No. 4,769,061 to Comai. Kumada et al. European patent application 0 333 033 to, and Goodman et al. U.S. Pat. No. 4,975,374 discloses a nucleotide sequence of a glutamine synthase gene that confers resistance to herbicides such as L-phosphinotricin. The nucleotide sequence of the PAT gene is described in Leemans et al. In European application 0 242 246. DeGreen et al. Bio / Technology 7:61 (1989) describe the production of transgenic plants that express a chimeric bar gene encoding for phosphinothricin acetyltransferase activity. Exemplary genes that confer resistance to phenoxypropionic acid and cyclohexane, such as cetoxydim and haloxyhops, are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described below: Marshall et al. , Theor. Appl. Genet. 83: 435 (1992).
C. Herbicides that inhibit photosynthesis, such as triazine (psbA and gs + genes) or benzonitrile (nitrilase gene) Przibila et al. , Plant Cell 3: 169 (1991) describes the transformation of Chlamydomonas with a plasmid encoding mutant psbA. Nucleotide sequences for nitrilase genes are disclosed in US Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genes are available under ATCC accession numbers 53435, 67441 and 67442. Cloning and expression of DNA encoding glutathione S-transferase is described below: Hayes et al. Biochem. J. et al. 285: 173 (1992).
D. Acetohydroxy acid synthase, which has been found to produce plants that express enzymes that are resistant to many types of herbicides, has been introduced into various plants. Hattori et al. Mol. Gen. Genet. 246: 419, 1995. Other genes that confer resistance to herbicides include genes encoding chimeric proteins of rat chromosome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., Plant Physiol., 106: 17, 1994), glutathione. Genes for reductase and superoxide dismutase (Aono et al., Plant Cell Physiol. 36: 1687, 1995), and genes for various phosphotransferases (Datta et al., Plant Mol. Biol. 20: 619, 1992) Is mentioned.
E. The protox enzyme, for which protoporphyrinogen oxidase (protox) is essential for the production of chlorophyll, which is necessary for the survival of all plants, serves as a target for various herbicidal compounds. These herbicides also inhibit the growth of all the different species of plants present, resulting in total destruction. The development of plants with modified protox activity that are resistant to these herbicides is described below: US Patents 6,288,306; 6,282,837; 5,767,373 and International Publication WO 01/12825.

3. A gene that imparts or contributes to a value-added trait. For example:
A. Modified fatty acid metabolism. For example, by transformation of a plant to increase the stearic acid content of the plant with an antisense gene for stearyl-ACP desaturase. Knultzon et al. , Proc. Natl. Acad. Sci. USA 89: 2625 (1992).
B. Reduced phytate content-1) Introduction of genes encoding phytases increases phytate degradation and adds more free phosphate to transformed plants. For example, for disclosure of the nucleotide sequence of the Aspergillus niger phytase gene, see Van Hartingsveldt et al. Gene 127: 87 (1993). 2) The gene could also be induced with reduced phytase content. In maize, for example, this could be achieved by cloning and then reintroducing DNA associated with one allele responsible for the maize mutation characterized by low levels of phytic acid. Raboy et al. , Maydica 35: 383 (1990).
C. Modified carbohydrate composition. For example, by transforming a plant with a gene encoding an enzyme that alters the branching pattern of starch. Shiroza et al. , J .; Bacteriol. 170: 810 (1988) (nucleotide sequence of Streptococcus mutated fructosyltransferase gene), Steinmetz et al. Mol. Gen. Genet. 20: 220 (1985) (Nucleotide sequence of the Bacillus subtilis levansucrase gene), Pen et al. Bio / Technology 10: 292 (1992) (production of transgenic plants expressing Bacillus lichenifonis α-amylase), Elliot et al. Plant Molec. Biol. 21: 515 (1993) (nucleotide sequence of the tomato invertase gene), Sogaard et al. , J .; Biol. Chem. 268: 22480 (1993) (site-directed mutagenesis of the barley α-amylase gene) and Fisher et al. , Plant Physiol. 102: 1045 (1993) (corn endosperm starch branching enzyme II).
D. Increase in oleic acid via FAD-2 gene modification and / or increase in linolenic acid via FAD-3 gene modification See US Pat. Nos. 6,063,947; 6,323,392; and International Publication WO 93/11245. I want to be.

4). Genes controlling male sterility A. Introduction of a deacetylase gene under the control of a tapetum specific promoter and addition of chemical N-Ac-PPT. See International Publication WO 01/29237.
B. Introduction of various stamen-specific promoters. See International Publications WO 92/13956 and WO 92/13957.
C. Introduction of barnase and barstar genes Paul et al. , Plant Mol. Biol. 19: 611-622, 1992.

Methods of Potato Transformation Many methods for plant transformation have been developed, including biological and physical plant transformation protocols. For example, Miki et al. , “Procedures for Introducing Forwarding DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B .; R. and Thompson, J.A. E. Eds. (CRC Press, Inc. Boca Raton, 1993) pages 67-88. In addition, vector expression and in vitro culture methods or tissue transformation and plant regeneration for plant cells are available. For example, Gruber et al. , “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick, B .; R. and Thompson, J.A. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

  A. Agrobacterium-mediated transformation—One method for introducing vectors into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al. , Science 227: 1229 (1985). A. Tumefaciens and A.E. Rhizogenes is a phytopathogenic soil bacterium that genetically transforms plant cells. A. Tumefaciens and A.E. Rhizogenes Ti and Ri plasmids each carry genes responsible for genetic transformation of plants. For example, Kado, C.I. I. Crit. Rev. Plant Sci. 10: 1 (1991). A description of the Agrobacterium vector system and methods for Agrobacterium-mediated gene transfer can be found in Gruber et al. , Supra, Miki et al. , Supra and Moloney et al. , Plant Cell Reports 8: 238 (1989). See also US Pat. No. 5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

  B. Direct gene transfer-Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as alternatives to Agrobacterium-mediated transformation. Genetically compatible method of 1-4 μm microinjection on the plant surface. The expression vector is introduced into the plant tissue by a particle gun device that accelerates microinjection to a speed of 300-600 m / sec sufficient to penetrate the plant walls and cell membranes. Sanford et al. , Part. Sci. Technol. 5:27 (1987); Sanford, J. et al. C. , Trends Biotech. 6: 299 (1988); Klein et al. Bio / Tech. 6: 559-563 (1988); Sanford, J. et al. C. Physiol Plant 7: 206 (1990); Klein et al. Biotechnology 10: 268 (1992). US Pat. No. 5,015,580 (Christou, et al.) Issued May 14, 1991, and US Pat. No. 5,322,783 (Tomes, et al.) Issued June 21, 1994. See also.

Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al. Bio / Technology 9: 996 (1991). Alternatively, liposome and spheroplast fusion have been used to introduce expression vectors into plants. Dehayes et al. , EMBO J. et al. 4: 2731 (1985); Christou et al. Proc Natl. Acad. Sci. USA 84: 3962 (1987). Direct incorporation of DNA into the protoplasm using CaCl ₂ precipitation, polyvinyl alcohol or poly-L-ornithine has also been reported. Hain et al. Mol. Gen. Genet. 199: 161 (1985) and Draper et al. , Plant Cell Physiol. 23: 451 (1982). Protoplasm and whole cell and tissue electroporation have also been described. Donn et al. , In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Hallin et al. Plant Cell 4: 1495-1505 (1992) and Spencer et al. , Plant Mol. Biol. 24: 51-61 (1994).

  Following transformation of the potato target tissue, the expression of the selectable marker gene described above allows for preferential selection of transformed cells, tissues and / or plants using regeneration and selection methods well known in the art.

  The methods described above for transformation are typically used to produce transgenic varieties. The transgenic variant is then crossed to another (non-transformed or transformed) variant to produce a new transgenic variant. Alternatively, a genetic trait that has been genetically manipulated in a particular potato line using the transformation techniques described above may be transferred to another line using conventional backcrossing techniques well known in the field of plant growth. For example, a backcross approach may be used to move the engineered trait from a public, non-elite variant to an elite variant, or from a variant that contains an exogenous gene in its genome. You may move to. As used herein, “mating” may simply refer to multiplying X by Y, or may refer to the backcross process, depending on the context.

  Those skilled in the art will recognize that, when the term potato plant is used in the context of the present invention, it is also a derivative variant that retains essentially distinguishable characteristics of E12, such as a genetically converted plant of this variant or incorporated therein. It is also understood to include transgenic derivatives having one or more added value genes (eg, herbicide or pest resistance). The backcross method can be used with the present invention to improve features or introduce features into variants. The term “backcross” as used herein refers to 1, 2, 3, 4, 5, 6, 7, 8, 9 or more repeated crossings that return the hybrid offspring to the regressed parent. A parent potato plant that has been subjected to a gene for one or more desired characteristics is referred to as a non-recurrent parent or donor parent. The term refers to the fact that non-regressing parents are used once in the backcross protocol and do not regress. A parent potato plant that has been transfected with a gene from a non-regressing parent is known as a recurrent parent and is used many times in the backcross protocol. In a typical backcross protocol, the original variant of interest (regressive parent) is crossed with a second variant (non-regressive parent) having the transferred gene of interest. The progeny resulting from this crossing is then again crossed to the regressing parent, and this process is added to the reverting parent's desired morphological and in addition to one or more genes transferred from the non-regressing parent in the transformed plant. Repeat until a potato plant is obtained that has essentially recovered all of its physiological characteristics.

  Selection of a suitable regression parent is an important step for a continuous backcross procedure. The goal of the backcross protocol is to modify one or more traits or features in the original variant. To accomplish this, one or more genes of the regression variant are modified, replaced, or supplemented with the desired gene from the non-regressive parent, while all the rest of the desired gene is essential. And thus the desired physiological and morphological structure of the original variant. The selection of a specific non-regressive parent depends on backcrossing. Adds agriculturally important traits to plants that are desirable in some markets. The exact backcross protocol will depend on the features or traits that have been modified or added to determine the appropriate test protocol. The backcross method is simplified if the feature to be transferred is a dominant allele, but a recessive allele can also be transferred. In this case, a progeny test needs to be introduced to determine if the desired feature has been successfully imported.

Thus, transgenes can be introduced using any of a variety of established and well-known recombinant methods. For example: Gressel, 1985, Biotechnologically Conferencing Herbicide Resistance in Crops: The Present Realities, In Molecular Form and Function of the Plant Gen. van Vloten-Dotting, (ed.), Plenum Press, New York; Hutner, S .; L. , Et al. 1992, Revising Oversight of Genetically Modified Plants, Bio / Technology; Klee, H .; , Et al. , 1989, Plant Gene Vectors and Genetic Transformation: Plant Transform Systems, Based on the use of Agrobacterium tumefaciens, Cell Culture and Sect. , Et al. , 1986, The Promoter of _TL -DNA Gene 5 Controls the Tissue-Specific Expression of Chimeric Gen and Carriage ByNel Type of Agrobacterium. , Et al. , 1990, Engineering Resistance to Mixed Virus Infection in a Commercial Potato Multival: Resistance to Potato Virus X and Potato Virus X and Potato Burst Y inTrak. A. , Et al. , 1996, Plants Resistant to Effect by PLRV. U.S. Pat. No. 5,510,253; Newell, C.I. A. , Et al. 1991, Agrobacterium-Mediated Transformation of Solanum tuberosum L .; Cv. Russet Burbank, Plant Cell Reports; J. et al. , Et al. , 1993, Genetically Improved Potatoes: Protection from Image by Color Colorado Potato Beetles, Plant Molecular Biology, all of which are incorporated herein by reference for this purpose.

  A number of traits have been identified that are not normally selected for the development of new variants, but can be improved by backcrossing and genetic engineering techniques. These traits may or may not be transgenic; examples of these traits include herbicide resistance; resistance to bacterial, fungal or viral diseases; insect resistance; starch or Uniformity or increase in the concentration of other carbohydrates; improved nutritional quality; reduced tendency of tubers to be damaged; and reduced rate of starch reduction to sugar. These genes are inherited genetically through the nucleus. Some of these traits are described below: US Patent 5,500,365, US Patent 5,387,756, US Patent 5,789,657, US Patent 5,503,999, US Patent 5, 589,612, US Pat. No. 5,510,253, US Pat. No. 5,304,730, US Pat. No. 5,382,429, US Pat. S. Patent N. US Pat. No. 5,503,999, US Pat. No. 5,648,249, US Pat. No. 5,312,912, US Pat. No. 5,498,533, US Pat. No. 5,276,268, US Pat. No. 4,900,676, US Pat. 633,434 and U.S. Pat. No. 4,970,168.

Deposit information R. The tuber deposit of potato cultivar E12, owned above and listed in the appended claims, owned by the Simple Company, was conducted by American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia. The date of deposit was May 23, 2013. Deposit 25 vials of microtubes in J. R. Received from the same deposit maintained by the Simple Company prior to the filing date of the present application. All restrictions are irreversibly removed at the time of grant of the patent, F. R. §§ 1.801 to 1.809 are intended to meet all requirements. The ATCC registration number is PTA-120372. The deposit will be maintained by the depositary authority over a period of 30 years, or 5 years from the last request, or for the longer the patentable photon period of the patent, if necessary, during that period, Be replaced.

  Although many exemplary aspects and embodiments are discussed above, those skilled in the art will recognize certain modifications, replacements, additions, and subcombinations thereof. Accordingly, the following appended claims are to be construed hereinafter as including all such modifications, replacements, additions, and subcombinations so as to be within the true spirit and scope thereof, Intended.

Claims (23)

  Potato tuber or tuber portion of potato cultivar E12, wherein a representative sample of said tubers is deposited under ATCC registration number PTA-120372, the portion of the potato tuber or tuber.   A potato plant or part thereof produced by growing the tuber or part of the tuber according to claim 1.   A potato plant having all of the physiological and morphological characteristics of the plant of claim 2.   A tissue culture of cells produced from the plant of claim 2, wherein the cells of the tissue culture are leaves, pollen, embryos, cotyledons, hypocotyls, meristem cells, roots, root canals, A tissue culture produced from a plant part selected from the group consisting of pistil, buds, flowers, stems and tubers.   5. A potato plant regenerated from the tissue culture of claim 4, wherein the plant has all the physiological and morphological characteristics of potato cultivar E12.   Potato seeds produced by growing the potato tubers or tuber parts of claim 1.   A potato plant or a part thereof produced by growing the seed according to claim 6.   A potato plant regenerated from a tissue culture of the potato plant according to claim 7, wherein the plant has all the physiological and morphological characteristics of potato cultivar E12.   A method for producing potato seeds, wherein at least one potato plant is crossed with two potato plants which are said potato plants according to claim 2 and harvesting the resulting potato seeds Including.   A method for producing potato seeds comprising crossing two potato plants, at least one potato plant being the potato plant of claim 7, and harvesting the resulting potato seeds. ,Method.   Potato seed produced by the method of claim 10.   A potato plant or a part thereof produced by growing the potato seed according to claim 11.   Potato seeds produced from the plant according to claim 12.   10. The method of claim 9, wherein one of the potato plants is transgenic and the other is potato cultivar E12.   11. The method of claim 10, wherein one of the potato plants is transgenic and the other is potato cultivar E12.   A potato plant or part thereof produced by the method of claim 14. A method for introducing a desired trait into potato cultivar E12, the method comprising:
(A) Producing progeny plants by crossing an E12 plant with a representative sample of tubers deposited under ATCC registration number PTA-120372 with another potato cultivar containing the desired trait. The desired trait is a group consisting of male sterility, herbicide resistance, insect resistance, modified fatty acid metabolism, modified carbohydrate metabolism and resistance to bacterial, fungal or viral diseases To be selected;
(B) selecting one or more progeny plants having the desired trait;
(C) backcrossing selected progeny plants with E12 plants to produce backcross progeny plants;
(D) selecting a backcross progeny plant having the desired trait; and (e) repeating steps (c) and (d) two or more times in succession to select a selected third containing the desired trait. Or producing more backcross progeny plants.   18. A potato plant produced by the method of claim 17, wherein the plant has the desired trait and has all the physiological and morphological characteristics of potato cultivar E12. .   The desired trait is herbicide tolerance, wherein the tolerance is conferred on a herbicide selected from the group consisting of imidazolinone, sulfonylurea, glufosinate, L-phosphinotricin, triazine and benzonitrile. Item 19. A potato plant according to item 18.   19. The potato plant of claim 18, wherein the desired trait is insect resistance, and the insect resistance is conferred by a transgene encoding a Bacillus thuringiensis endotoxin.   The desired trait is a modified fatty acid metabolism or a modified carbohydrate metabolism, wherein the desired trait is selected from the group consisting of fructosyltransferase, levansucrase, α-amylase, invertase and starch branching enzyme 19. A potato plant according to claim 18, which is conferred by a nucleic acid encoding a protein or a DNA encoding an antisense of stearyl-ACP desaturase.   A method of producing a commercial plant product comprising obtaining the plant or part thereof of claim 2 and producing a commercial plant product from the plant or plant part thereof, wherein the commercial plant product comprises fried food A method selected from the group consisting of potato, potato chips, dehydrated potato material, potato flakes and potato granules.   23. A commercial plant product produced by the method of claim 22.